Compositions and methods for treatment of microbial infections

ABSTRACT

The present invention relates to methods and compositions for treatment of microbial infections and for the enhancement of resistance to infection. The invention comprises administration of an effective amount of bacterial lysate compositions for the treatment of pathological conditions of microbial infections. The present invention can also be used to enhance the immune system to prevent infections by the administration of an effective amount of the compositions.

RELATED APPLICATION

This application claims benefit of U.S. Provisional Application No.60/356,483 filed Feb. 13, 2002, which is incorporated herein in itsentirety.

FIELD OF THE INVENTION

The present invention relates generally to the treatment of microbialinfections. More specifically, the invention relates to methods andcompositions for the enhancement of an immune response in a human oranimal to microbial infections.

BACKGROUND OF THE INVENTION

The idea of using phages for treatment of infectious diseases wasinitially proposed by d'Herelle in 1917. In the 1920s, the discovery ofbacteriophages was thought to be the answer for treatment of bacterialinfections. Bacteriophages invade and destroy bacteria and appeared tobe the selective therapeutic bullet that would knock out bacteriainvading an animal or plant host. Unfortunately, the phages were not aseffective in killing bacteria in host organisms as they were in killingbacteria in vitro. The development of antibiotics in the 1940s as thepreferred treatment for bacterial infections led to a decline inresearch into use of bacteriophages for treatments of infectiousdiseases.

In a few countries, notably in Eastern Europe and India, researchcontinued in the use of bacteriophages and bacterial lysates fortreatment of infectious diseases and enhancement of immune responses.Though much of that research is still not accepted widely in otherindustrialized countries, a renewed interest in this research is growingas a result of the occurrence of antibiotic resistant bacteria. Manyinfectious diseases that were once easily treatable with antibiotics arenow a serious health threat because the bacteria are resistant to most,if not all, antibiotics. Medical science is searching for treatmentsthat can respond to these resistant bacteria in a way that does not leadto even more resistant infectious strains.

The U.S. Centers for Disease Control and Prevention estimates that 20%to 50% of prescribed antibiotics are unnecessary. The overuse ofantibiotics in medicine and agriculture has greatly fostered thedevelopment of resistant strains of bacteria. In a microenvironmentbombarded with antibiotics, the few bacteria that can resist the drugsproliferate. Bacteria are becoming increasingly resistant to what werepreviously considered “last resort” antibiotics.

Bacteriophage therapy has not gained much attention or acceptance inmost industrialized countries, in part due to the reliance onpharmaceuticals, such as antibiotic treatment for bacterial infections.With the advent of increased bacterial resistance, bacteriophage therapyhas come under renewed scrutiny as a possible alternative topharmaceutical treatments of bacterial infections. What is needed arecompositions and methods for treatment and prevention of microbialinfections that do not rely on pharmaceutical antibiotic therapies. Suchmethods and compositions should be capable of treating or preventinginfections in organisms, including humans, animals and plants.

SUMMARY OF THE INVENTION

The present invention is directed towards compositions and methods oftreatment and prevention of microbial infections. Preferred methodsinclude administration of compositions comprising bacterial lysates. Thepresent invention contemplates use of any bacterium that has stablereproduction and does not produce a toxin or have deleterious effectseither on the bacteriophage or on the human or animal receiving theresulting lysate. Preferred bacterial lysates include, but are notlimited to, those derived from strains of Staphylococcus aureus (S.aureus), Klebsiella pneumoniae, (K. pneumoniae) and Pseudomonasaeruginosa (P. aeruginosa). Most preferred bacterial strains comprisebacterial strains deposited at the Czech Collection of Microorganisms(CCM) having accession numbers, CCM 4992, CCM 4993, CCM 4994, CCM 4995,CCM 4996, CCM 4997, CCM 4998. Most preferred bacteriophages comprisebacteriophages deposited at the Deutsche Sammlung von Mikroorganismenund Zellkulturen GmbH (DSM) and having accession numbers, 14614, 14615,and 14616.

The present invention comprises a composition, comprising a bacteriallysate derived from the infection of at least one bacterial strain of S.aureus, K. pneumoniae, or P. aeruginosa with a bacteriophage. Thecomposition of the present invention further comprises a bacteriallysate derived from one or more S. aureus bacterial strains depositedwith the CCM under accession numbers CCM 4992, CCM 4993, CCM 4994, CCM4995, CCM 4996, CCM 4997, and CCM 4998. The present invention alsocomprises a bacterial lysate derived from bacteriophages deposited withthe DSM having accession numbers DSM 14614, DSM 14615, and DSM 14616.The present invention additionally comprises S. aureus bacterialisolates deposited with the CCM under accession numbers CCM 4992, CCM4993, CCM 4994, CCM 4995, CCM 4996, CCM 4997, and CCM 4998. The presentinvention also comprises bacteriophages, deposited with the DSM underaccession number DSM 14614, DSM 14615, and DSM 14616.

The bacterial lysates of the present invention may be used in methods oftreating or preventing pathological conditions of microbial infectionsin humans or animals. The compositions and inventions of the presentinvention may further comprise a suitable pharmaceutical excipient. Thepathological conditions of microbial infections which can be treated orprevented by the present invention include, but are not limited to,conditions such as chronic upper respiratory disease, wound infection,osteomyelitis, endocarditis, skin polymicrobial infections, bronchialasthma, chronic sinusitis, cystic fibrosis or acne vulgaris.

The compositions of the present invention may be used as vaccinecompositions as well as in the treatment specific microbial infections.The compositions of the present invention may consist of one or morebacterial lysates formed by infecting specific strains of bacteria withbacteriophages. The compositions can be used in methods for thetreatment and prevention of local or systemic bacterial infectionsincluding, but not limited to, chronic or recurrent respiratory or earinfections, post operational infections, bacterial pneumonia infections,sepsis, skin infections, wound infection, osteomyelitis, allergies,asthma, sinusitis, and acne vulgaris. Additionally, the compositions ofthe present invention may be used in methods for immune stimulation.

DETAILED DESCRIPTION OF THE INVENTION

The present invention comprises compositions and methods for thetreatment of microbial infections comprising administering an effectiveamount of one or more bacterial lysates or mixtures thereof. Theimmunogenic compositions of the present invention may further compriseat least one or more immunogenic or immunostimulating materials orformulations for regulating or affecting microbial distribution in anorganism.

The present invention also comprises compositions and methods ofvaccination against microbial infections comprising administeringcompositions comprising one or more bacterial lysates or mixturesthereof and a pharmaceutically acceptable carrier. The lysates may beused individually or in combination. The vaccines of the presentinvention are used to immunize animals, including humans, againstbacterial diseases by administering to the human or animal an effectiveimmunizing amount of the bacterial lysate.

The compositions of the present invention comprise bacterial lysatesproduced by infecting selected bacteria with selected bacteriophages.The present invention contemplates use of any bacteriophage that stablyreproduces in a selected bacterial strain and does not result in theproduction of a significant amount of a toxin or another component thathas deleterious effects either on the host bacteria or in the resultinglysate that is deleterious to the human or animal receiving the lysate.Determination and selection of such bacteriophages are performed usingtechniques known to microbiologists and include testing for long termstability in a general host strain, long term persistence of virulence,stability and reproducibility of lysis and reproducibility of resultantlysates. The host strain is a strain which is extremely sensitive to thelytic properties of the bacteriophage. It is selected from the strainssensitive to the particular bacteriophage.

The compositions of the present invention further comprise bacteriallysates derived from selected bacteria. The present inventioncontemplates use of any bacterium that has stable reproduction and doesnot produce a toxin that has deleterious effects either on thebacteriophage or in the lysate recipient. Determination and selection ofsuch bacteria is performed using techniques known to those skilled inthe art and includes testing for the absence of toxins particularly:alpha toxin, beta toxin, delta toxin, gamma toxin, enterotoxins A, B, C,and D, Toxic Shock Syndrome Toxin (TSST), exfoliatins A and B,leukocidin, fatty acid modifying enzyme, and hemolysins. Testing alsoincludes tests for stability and reproducibility of lysates generated bythe phage-induced lysis, long-term sensitivity of the bacterial strains,and continued susceptibility of the bacteria to lysis by phages withoutthe appearance of phage-resistant colonies.

Selection of a lysate composition is determined by the methods of usefor a particular lysate composition. For example, if the desired use isto provide immunity for staphylococcal infections, one or more strainsof staphylococcal bacteria are used as the bacterial host organisms. Inthis same example, one or more bacteriophages that are specific forstaphylococcal bacteria, or are at least capable of having a productiveinfection in staphylococcal bacteria, are used to create thestaphylococcal lysate. Alternatively, one bacterial strain may be grownto produce a bacterial culture or bacterial broth and then separatealiquots of the bacterial culture are each infected with a differentbacteriophage to create individual lysates. These individual lysates maybe used individually or combined to form compositions. In anotherembodiment, different bacterial strains are grown and then each isinfected with the same bacteriophage to yield lysate compositions thatcan be used individually or combined to form compositions. Anotherembodiment of the present invention contemplates the use of differentbacterial strains that are each infected with different bacteriophagesand the resulting lysates are used individually or in combination toform compositions.

The compositions of the present invention preferably comprisecompositions comprising lysates from at least one bacterial strain, morepreferably, two or more bacterial strains A preferred compositioncomprises lysates from one of the following bacteria: Staphylococcusaureus (S. aureus), Klebsiella pneumoniae (K. pneumoniae), andPseudomonas aeruginosa (P. aeruginosa). Preferred compositions compriselysates derived from bacterial strains of S. aureus deposited with theCzech Collection of Microorganisms (CCM) on Oct. 11, 2001 and havingaccession nos. CCM 4992, CCM 4993, CCM 4994, CCM 4995, CCM 4996, CCM4997 and CCM 4998. Preferred compositions additionally comprise thebacterial strains deposited with the CCM on Oct. 11, 2001 and havingaccession numbers CCM 4992, CCM 4993, CCM 4994, CCM 4995, CCM 4996, CCM4997 and CCM 4998

The methods of the present invention comprise the use of bacteriophagesto yield the desired bacteriophage lysates. A more preferred methodcomprises lysates produced by bacteriophages for S. aureus, K.pneumoniae, and P. aeruginosa. Most preferred methods comprise lysatesderived from bacteriophages deposited on Nov. 19, 2001 with the DeutscheSammlung von Mikroorganismen and Zellkulturen GmbH (DSM) and havingaccession numbers 14614, 14615, and 14616. Preferred compositionsadditionally comprise the bacteriophages deposited on Nov. 19, 2001 withthe Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSM)and having accession numbers 14614, 14615, and 14616. The bacteriophagescan be used alone or in combination to lyse one or more differentbacterial strains.

Generally, the methods of the present invention comprise growing one ormore desired bacterial strains to a desired concentration. For example,ranges of preferred concentrations of bacterial cultures are from 1×10⁶to 1×10¹² cells/ml, preferably 1×10⁷ cells/ml, 1×10⁸ cells/ml, 1×10⁹cells/ml, 1×10¹⁰ cells/ml, 1×10¹¹ cells/mi. At the selected time, one ormore bacteriophages, at an effective concentration, are added to thebacterial culture. For example, ranges of preferred concentrations ofbacterial phage cultures are from 1×10⁸ to 1×10¹², preferably, 1×10⁹,1×10¹⁰, 1×10¹¹. Infection and lysis by the bacteriophages is allowed toproceed. Incubation of the bacteriophage or phages with the bacterialculture can be from 3 to 48 hours. The resulting bacterial lysate isthen filter sterilized using filters having a pore size from <0.1 μm to0.45 μm.

Both the material retained on the filter, the rententate, and the flowthrough material, the filtrate, are kept. It is preferred to use thefiltrate for compositions in methods of treatment and immunestimulation. In general, the filtrate comprises cellular materials suchas components of cell walls, cellular membranes, proteins, ribosomalfractions, glycoproteins, DNA, RNA, and the like. The rententate fromultrafiltration using filters with a pore size of <0.1 micrometerscomprises larger cellular materials and unlysed cells and bacteriophageswhich may also be used in the compositions and methods of the presentinvention.

The use of bacteriophages to prepare lysates is effective for all typesof bacteria and allows the preparation of lysates to evolve with thebacteria and avoid resistance problems common to pharmaceuticals. Theuse of bacteriophage lysates allows the preparation of products capableof modulating the immune system prepared from a single bacterial strain,or a combination of bacterial strains.

In general, methods of the present invention comprise screening strainsof a particular bacterial family or group that does not produce toxins,for selected bacteria that exhibit the most common antigenic patternfound in the bacterial family or group. The antigenic pattern can bedetermined by immunogenic methods such as fluorescent antibodyidentification, FACS selection, ELISA, Western blots, SDS gelelectrophoresis or by other detection methods known to those skilled inthe art. Cultures of the selected bacteria are then infected by theintroduction of phage homologues. The combination is screened forstrains that result in quantitative lysis of bacteria in a given timeand strains that exhibit the most common antigenic pattern. Thebacterial strain and phage are then preserved, preferably bylyophilization, to maintain stability. The stability of the bacterialstrains and phages are determined through stability assays, repeatedcultures and subcultures on media. The bacterial strains and phages arealso checked for antigenic infection and reproducibility of antigenicpatterns of lysates. At least one, preferably two or more, of theresultant lysates are combined to yield an immunomodulator composition.Therefore, the resulting lysate from the lysis can be quantitatively andqualitatively analyzed by SDS gel electrophoresis.

The compositions can be used in methods for the treatment of local orsystemic bacterial infections including, but not limited to, chronic orrecurrent respiratory or ear infections, prophylactic prevention ofinfections and treatment of pathological conditions of microbialinfections such as post operational infections, bacterial pneumoniainfections, sepsis, skin infections, wound infection, osteomyelitis,skin polymicrobial infections, allergies, asthma, endocarditis,arthritis, abscess, sinusitis, and acne vulgaris. Additionally,bacteriophage lysates can be used in methods for immune stimulation suchas vaccines that boost the immune system. The compositions can also beadministered for several months prior to planned operations such as kneeor hip replacements to boost general immune response, decrease recoverytime, and prevent nosocomial infections.

Methods of treatment contemplated by the present invention compriseadministration of an effective amount of a bacterial lysate compositionto an organism having an infection or in need of immune stimulation. Aneffective amount of a bacterial lysate composition can be determined byusing known amounts of such compositions, or by starting treatment witha small dose and increasing the dose until the desired effect isachieved. Such techniques for determining effective amounts are known tothose skilled in the art and do not require undue experimentation todetermine. Differing routes of administration including, but not limitedto, oral, buccal, nasal, aerosol, topical, transdermal, injectable, slowrelease, controlled release, iontophoresis, sonophoresis, intravenous,intramuscular, intraperitoneal, intraspinal, intrathecal,intracerebroventricular, intraarterial, subcutaneous and intranasalroutes, generally require differing effective amounts to achieve thedesired result. For example, oral administration may require differenteffective amounts than topically administered effective amounts. Ingeneral, a range of 0.0001 mg/kg/day to 500 mg/kg/day provides aneffective amount of a bacterial lysate composition.

Preferred methods comprise administration of the lysate three to sixweeks prior to reconstructive surgery such as, but not limited to, aknee or hip replacement. For example, the lysate is administered in adose is 0.05 cc, injected intradermally twice a week. Three days afteradministration of the initial dose, the patient is observed fortolerance of the lysate. If no local reaction is noted, the dose can beincreased to 0.1 cc up to 0.2 cc. The lysate is then administered everyother day. In the case of nasal routes of administration, preferably twoto four drops are placed in each nostril the first four days and afterfour days, the dosage is repeated every other day. The length oftreatment is generally three to four months. Multiple methods ofadministration may be combined with the length of treatment depending onthe immune status of the patient.

The present invention further comprises methods of eliciting an immuneresponse in a host comprising administering an effective amount of animmunogenic composition. The immunogenic composition may be usedprophylactically as part of a vaccination system in which thecomposition is administered prior to infection, or in the treatment of aparticular infection. The immune response may be a humoral or acell-mediated immune response. Immunogenicity may be improved theco-administration of antigens with adjuvants. Adjuvants may act byretaining the antigen locally near the site of administrationfacilitating a slow sustained release of antigen to cells. Adjuvants canalso attract immune cells to the site of injection and stimulate suchcells to elicit immune responses. A wide range of adjuvants can aid inevoking an immune responses. These include, but are not limited to,pluronic polymers with mineral oil, Freund's complete adjuvant, lipid A,liposomes and cholera toxin subunit B or its genetically modifiedvariants.

The following specific examples of the methods and compositions of thepresent invention are in no way to be seen as limiting, but merelyprovide illustrative embodiments for the applications of the presentinvention. The present invention contemplates the use of bacteriallysates for the treatment of bacterial and other microbial infectionsand for stimulation of the immune system in general and other diseasetreatments or compositions are not limited by the specific examplestaught herein.

Staphylococcus aureus Infections Prevention and Treatment

Staphylococcal bacteria, and S. aureus in particular, are some of themore common and virulent pathogens. Some staphylococcal infections arecharacterized by intense suppuration, necrosis of local tissues and theformation of abscesses. Staphylococcal infections are responsible forskin infections, such as furuncles, carbuncles and impetigo, and deeplesions spreading from the skin into bones, joints, soft tissues andorgans. S. aureus can produce toxins leading to scalded skin syndrome,toxic shock syndrome and staphylococcal food poisoning. It is a majorcause of wound infection and a continuing cause of hospital epidemics.S. aureus is currently resistant to many classes of antibiotics and iscurrently being treated with vancomycin as a last resort. However,strains resistant to vancomycin have already begun to appear.

S. aureus has many virulence factors that act against native or adaptiveimmune responses. While not wishing to be bound by any particulartheory, it is currently believed that part of the innate responseagainst S. aureus comprises the Toll-like receptor (TLR) family. TLRsare transmembrane receptors linking the pattern recognition system ofpathogens with intracellular signaling. In humans, the TLR family isrepresented by TLR (TLR 1-10), several proteins related to TLR (RP105,Nod1, Nod2), as well as lipopolysaccharide (LPS) receptor (CD14).Several TLRs appear to have a specific function in the patternrecognition system. For example, TLR-2 is believed to activate cells inresponse to gram-positive cell-wall components (peptidoglycan). TLR-4 isimportant (in complex with CD14) for recognition of LPS (gram-negativebacteria). TLR-9 recognizes CpG motif of bacterial DNA. The activationof the innate immune system induces early inflammatory reaction mediatedby monocytes, neutrophils, and endothelial cells. The response can occurwith or without participation of the adaptive immune system, leading toleukocyte recruitment, production of pro-inflammatory cytokines(TNF-alpha, IL-1 beta), reactive oxygen products, and effector cytokines(e.g., IL-12) acting on T cells, antigen presenting cells, and by acascade of cytokines indirectly on B cells.

Mice deficient in TLR-2 or a signaling molecule (MyD88) were highlysusceptible to S. aureus infection (J. Immunol. 165, 5392-5396, 2000).Furthermore, wild-type mice became more resistant to acute polymicrobialsepsis after treatment with CpG DNA (J. Immunol. 165, 4537-4543, 2000)that is currently believed to act via TLR-9. TLR-2 is likely to play animportant role in human S. aureus infection as evidenced by detection ofTLR-2 polymorphism associated with S. aureus septic shock (Infect.Immun. 68, 6398, 2000). Activation of TLR-2 receptor with S. aureus cellwall components results in upregulation of Th-1 pathway (TNF-alpha,interpheron gamma, IL-1, etc.). Increase of these pro-inflammatorycytokines activates and modulates multiple responses of innate andadaptive immunity.

S. aureus has several mechanisms to interfere with the normal immuneresponse. Not only has the bacteria developed a resistance againstantimicrobial proteins but it can also survive inside neutrophils. Inaddition, various strains of S. aureus possess some of the battery oftoxins and enzymes and other products that enhance infectivity andbacterial survival and proliferation. Antibodies specific against toxinsand enzymes and other soluble products neutralize the activities of thetoxins while antibodies specific against cells and cellular componentsopsonize the targeted cells and enhance their phagocytosis.

Treatment of staphylococcal infections and S. aureus in particular, areprovided by the present invention. Selected staphylococcal lysatecompositions comprising lysate from single or multiple strains ofstaphylococcus and bacteriophages are administered through variousroutes, including but not limited to, nasal drops and subcutaneousroutes, or a combination thereof. Lysates from different strains ofstaphylococcus or lysates that result from different bacterial phageinoculations are preferably combined in equal proportions.Staphyloccocal lysate (SPL) has been clinically effective in thetreatment of all staphylococcal infections of adults, as well as chronicconditions in pediatrics (chronic upper respiratory diseases, bronchialasthma, chronic sinusitis, cystic fibrosis) and in dermatology (acnevulgaris).

SPL is prepared by lysis of S. aureus culture with a polyvalentbacteriophage. Most staphylococcal phages belong to the Siphoviridaefamily, e.g., phages with double-stranded linear DNA and with long,noncontractile tails. SPL is a complex of antigenic components ofribosomal, cytoplasmic, nuclear, cell wall, and membranous origin.Purified components of SPL can be used for specific stimulation ofcertain pathways including, but not limited to, peptidoglycans viaTLR-2, CpG DNA via TLR-9.

Alternatively, animals and humans can be vaccinated with SPLcompositions so that staphylococcal infections are resisted. Suchtreatment comprises administration of a SPL composition comprising lysedbacteria and phage through routes of administration comprisingsubcutaneous injections, nasal drop application and lavage ofosteomyelitis fistulas. The administration of the lysate compositionscan take place prophylacticly, prior to the occurrence of astaphylococcal infection, as well as during a staphylococcal infection.Preferred times of prophylactic administration are after the age ofthree for three to six weeks prior to potential infections situations orgenerally as part of a vaccination program.

Klebsiella pneumoniae Infection Prevention and Treatment

Another bacteria group for which the present invention is effective isthe Klebsiella bacteria, in particular, K pneumoniae. K. pneumoniae is anonopportunistic pathogen normally found in the gut. Outside of the gut,it is a growing source of hospital-acquired infections causing pneumoniaand urinary tract infections. Patients with chronic respiratorydiseases, diabetics, alcoholics, and people of advanced age, as well asthose using respiratory therapy devices, intravenous and urinarycatheters are particularly sensitive to acquiring infectious Klebsiella.

Classically, K. pneumoniae infection of the respiratory tract causes asevere, rapid-onset illness that often results in destruction of areasin the lung. Even with treatment, the mortality rate due to K.pneumoniae is 50%. Infected persons generally develop high fever,chills, flu-like symptoms and a cough that produces a lot of mucous.While normal bacterial pneumonia frequently resolves withoutcomplication, K. pneumoniae frequently causes lung destruction andabscesses.

Klebsiella can also cause less serious respiratory infections, such asbronchitis, which is usually a hospital-acquired infection. Other commonhospital-acquired infections caused by Klebsiella are urinary tractinfections, surgical wound infections and bacteremia. All of theseinfections can progress to shock and death if not treated early in anaggressive fashion.

Methods of treatment of Klebsiella infections and K. pneumoniae inparticular, are provided by the present invention. Selected klebsiellalysate compositions comprising single or multiple strains of klebsiellaor bacteriophages are administered through various routes, including butnot limited to, nasal drops and subcutaneous routes, or a combinationthereof. The lysate compositions are administered daily until thedesired treatment is achieved.

Alternatively, animals and humans can be vaccinated or treatedprophylactically with bacterial lysate compositions so that klebsiellainfections are resisted. Such treatment comprises administration ofklebsiella lysate compositions comprising lysates from single ormultiple strains of klebsiella and phage through routes ofadministration comprising nasal drops and subcutaneous administration attimes prior to infection with klebsiella. Preferred prophylacticadministration for humans is after the age of 3, for 3-6 weeks prior topotential infectious conditions such as hospitalizations or generally aspart of a vaccination program.

Pseudomonas aeruginosa Infection Prevention and Treatment

Another frequent hospital contaminant, P. aeruginosa is a versatile,gram-negative bacterium that grows in soil, marshes, and coastal marinehabitats, as well as on plant and animal tissues. P. aeruginosa is ableto grow in aqueous solutions including distilled water. This abilityresults in frequent contamination of i.v. fluids, respirators,anesthesia equipment and other hospital equipment. People with cysticfibrosis, burn victims, individuals with cancer, and patients requiringextensive stays in intensive care units are particularly at risk of P.aeruginosa infection. Unlike many environmental bacteria, P. aeruginosahas a remarkable capacity to cause disease in susceptible hosts. It hasthe ability to adapt to and thrive in many ecological niches, from waterand soil to plant and animal tissues. P. aeruginosa can produce a numberof toxic proteins which not only cause extensive tissue damage, but alsointerfere with the immune system's defense mechanisms. These proteinsrange from potent toxins that enter and kill host cells at or near thesite of colonization to degradative enzymes that permanently disrupt thecell membranes and connective tissues in various organs.

P. aeruginosa infections are a particular problem in individuals withcystic fibrosis. Cystic fibrosis is a common lethal genetic disease(incidence 1:2,000 Caucasian births). The disease presents with ahistory of chronic lung disease, recurrent pneumonia, cough, andbronchiectasis. The lungs of cystic fibrosis patients may be colonizedwith P. aeruginosa, particularly a special mucoid strain thatcontributes to fatal complications. S. aureus is frequently present aswell.

Treatment of pseudomonas infections and P. aeroginosa in particular, areprovided by the present invention. Selected pseudomonas lysatecompositions comprising lysates from single or multiple strains areadministered through various routes, including but not limited to, nasaldrops and subcutaneous routes, or a combination thereof.

Alternatively, animals and humans can be vaccinated with pseudomonaslysate compositions so that pseudomonas and pneumococcal infections areresisted. Such treatment comprises administration of pseudomonas lysatecomposition comprising lysates from bacteria and phage through routes ofadministration comprising nasal and subcutaneous injection at timesprior to infection with pseudomonas. Preferred times of preventativeadministration are after 3 years of age, for three to six weeks prior tothe potential infectious situations such as proposed hospitalization orgenerally as part of a vaccination program.

Mastitis

The present invention is effective for the prevention and treatment ofmastitis, particularly mastitis in dairy cattle, though any mastitis canbe treated using the present invention. Mastitis in dairy cattle is aninflammation of the mammary gland in response to intramammary bacterialinfection, mechanical trauma, or chemical trauma. Economic losses due tomastitis are 51.7 billion dollars a year in the U.S. alone. It isthought that contagious mastitis is primarily caused by S. aureus andStreptococcal agalactiae. Environmental mastitis can be caused by avariety of different bacteria, including, but not limited to, K.pneumoniae, Escherichia coli, Klebsiella oxytoca, Enterobacteraerogenes, Streptococcal uberis, Streptococcal bovis, and Streptococcaldysgalactia.

Traditional prevention of bovine mastitis involves a complex regimen ofdaily teat-dipping with a disinfectant solution, and may involveantibiotic-containing teat dips. When infection does occur, intramammaryinfusion of antibiotics is indicated, however this leads to increasinglyresistant strains of bacteria. Antibiotic therapy can reduce theinfection so that the milk produced is saleable, but it generally doesnot lead to complete elimination of the causative organism. While notwishing to be bound to any particular theory, studies on mastitis haveindicated that part of the problem in treating mastitis is that asignificant number of bacteria remain viable in the mammary gland withinphagocytic polymorphonuclear neutrophil leukocytes (PMN). When lysis ofthe leukocyte occurs, the phagocytized bacteria may provide a renewedsource of mastitis producing, for example, staphylococcal regrowth.While not wishing to be bound, it is currently believed that the use ofbacterial lysates allows for the creation of memory cells, allowing thecow to respond to remaining bacteria, whenever they might appear.

In general, bovine mastitis is treated by administering an effectiveamount of a bacterial lysate to a cow. The administration may be aprophylactic administration, in that all cattle in the herd are treatedwith bacterial lysate compositions, or the administration may occur wheninfection occurs in individual cows. For example, in heifers,vaccination starts at 6 months of age. Three subcutaneous injections oflysate (5 cc each) are applied between 6 and 24 months of age.

The present invention is also useful in the treatment of skin infectionsin other domesticated animals including canine and feline staphylococcalskin infection, dermatitis, and other chronic infections. Treatmentmethods comprise the administration of an effective amount of abacterial lysate to domesticated animals.

Bacterial antigens are known to trigger immunomodulatory effects invivo. For example, in an infection of a host by a staphylococcalorganism, other bacteria and some viruses, it is believed that astaphylococcus lysate composition potentiates cell-mediated as well ashumoral immunity in animals and humans. Treatment with staphylococcuslysates elevates antigen specific as well as total humoral antibody andimmunoglobulin levels creating an effective immunoadjuvant for humoralresponses as well.

Delivery and Dosage

The methods of the present invention comprise routes of administrationthat include, but are not limited to, oral, buccal, nasal, aerosol,topical, transdermal, injectable, slow release, controlled release,iontophoresis, sonophoresis, and other delivery devices and methods.Injectable methods include, but are not limited to, intravenous,intramuscular, intraperitoneal, intraspinal, intrathecal,intracerebroventricular, intraarterial, subcutaneous and intranasalroutes.

The compositions for treating the pathologies by the present inventioncan further include a pharmaceutically acceptable carrier or excipient.The compositions can also include other medicinal agents, pharmaceuticalagents, carriers, adjuvants diluents and other pharmaceuticalpreparations known to those skilled in the art. These agents are knownto those skilled in the art and are generally described as beingbiologically inactive and can be administered to patients withoutcausing deleterious interactions with the active agent.

According to the invention, at least one pharmaceutical composition canbe delivered by any of a variety of inhalation or nasal devices known inthe art for administration of a therapeutic agent by inhalation. Devicescapable of depositing aerosolized formulations in the sinus cavity oralveoli of a patient include metered dose inhalers, nebulizers, drypowder generators, sprayers, and the like. Other devices suitable fordirecting pulmonary or nasal administration are also known in the art.

There are a several desirable features of an inhalation device foradministering a compound of the present invention. For example, deliveryby the inhalation device is advantageously reliable, reproducible, andaccurate. For pulmonary administration, at least one pharmaceuticalcomposition is delivered in a particle size effective for reaching thelower airways of the lung or sinuses.

All such inhalation devices be used for the administration of apharmaceutical composition in an aerosol. Such aerosols may compriseeither solutions (both aqueous and non aqueous) or solid particles.Metered dose inhalers like the Ventolin® metered dose inhaler, typicallyuse a propellent gas and require actuation during inspiration. See,e.g., WO 98/35888; WO 94/16970. Dry powder inhalers like Turbuhaler®(Astra), Rotahaler® (Glaxo), Diskus® (Glaxo), Spiros® inhaler (Dura),devices marketed by Inhale Therapeutics, and the Spinhaler powderinhaler (Fisons), use breath-actuation of a mixed powder. See U.S. Pat.Nos. 5,458,135; 4,668,218; WO 97/25086; WO 94/08552; WO 94/06498; and EP0 237 507, each entirely expressly incorporated herein by reference.Nebulizers like AERx®, Aradigm, the Ultravent® nebulizer (Mallinckrodt),and the Acorn II® nebulizer (Marquest Medical Products), the abovereferences entirely expressly incorporated herein by reference, produceaerosols from solutions, while metered dose inhalers, dry powderinhalers, etc. generate small particle aerosols. These specific examplesof commercially available inhalation devices are intended to be arepresentative of specific devices suitable for the practice of theinvention, and are not intended as limiting the scope of the invention.

Suitable formulations, wherein the carrier is a liquid, foradministration, as for example, a nasal spray or as nasal drops, includeaqueous or oily solutions of the active ingredient.

A spray comprising a pharmaceutical composition of the present inventioncan be produced by forcing a suspension or solution of a compounddisclosed herein through a nozzle under pressure. The nozzle size andconfiguration, the applied pressure, and the liquid feed rate can bechosen to achieve the desired output and particle size. An electrospraycan be produced, for example, by an electric field in connection with acapillary or nozzle feed.

A pharmaceutical composition of the present invention can beadministered by a nebulizer such as a jet nebulizer or an ultrasonicnebulizer. Typically, in a jet nebulizer, a compressed air source isused to create a high-velocity air jet through an orifice. As the gasexpands beyond the nozzle, a low-pressure region is created, which drawsa solution of composition protein through a capillary tube connected toa liquid reservoir. The liquid stream from the capillary tube is shearedinto unstable filaments and droplets as it exits the tube, creating theaerosol A range of configurations, flow rates, and baffle types can beemployed to achieve the desired performance characteristics from a givenjet nebulizer. In an ultrasonic nebulizer, high-frequency electricalenergy is used to create vibrational, mechanical energy, typicallyemploying a piezoelectric transducer. This energy is transmitted to theformulation of composition protein either directly or through a couplingfluid, creating an aerosol including the composition protein.

In a metered dose inhaler (MDI), a propellant, a compound of the presentinvention, and any excipients or other additives are contained in acanister as a mixture including a liquefied, compressed gas. Actuationof the metering valve releases the mixture as an aerosol.

Pharmaceutical compositions for use with a metered-dose inhaler devicewill generally include a finely divided powder containing a compounddisclosed herein as a suspension in a non-aqueous medium, for example,suspended in a propellant with the aid of a surfactant. The propellantcan be any conventional material employed for this purpose such aschlorofluorocarbon, a hydrochlorofluorocarbon, a hydrofluorocarbon, or ahydrocarbon including trichlorofluoromethane, dichlorodifluoromethane,dichlorotetrafluoroethanol and 1,1,1,2-tetrafluoroethane, HFA-134a(hydrofluroalkane-134a), HFA-227 (hydrofluroalkane-227), or the like.One of ordinary skill in the art will recognize that the methods of thepresent invention can be achieved by pulmonary administration of acompound disclosed herein via devices not described herein.

Simple lysate delivery systems of the present invention comprisecapsules containing differently coated pellets of the lysate. On releasefrom the capsule, the uncoated pellets provide an initial amount of thelysate composition to the body, and the coated pellets provide thelysate composition over a period of time. Another system includeshydrogel materials with coated pills embedded in the hydrogel, such asthat taught in U.S. Pat. No. 4,659,558. The unswollen hydrogel, such asthat taught in U.S. Pat. No. 4,659,558, is swallowed and in the presenceof fluids in the stomach, swells so that the hydrogel is retained withinthe stomach. The coated pills are released as the hydrogel degrades. Thelysate may also be administered through the use of auto-injectiondevices such as those described in U.S. Pat. Nos. 5,514,097; 159,192;and 5,643,214; as well as European Patent No. 0 516 473 B1.

In controlled release systems contemplated in the present invention,after oral ingestion, lysates are released by diffusion and erosionthroughout the gastrointestinal tract to a significant degree. Methodsof the present invention for the prolongation of gastric retention timeinclude incorporation of fatty acids to reduce physiological gastricemptying and the use of bioadhesive polymers. Such systems are known tothose skilled in the art and comprise using polymers such aspolycarbophyll, sodium carboxymethylcellulose, tragacanth gum, acrylatesand methacrylates, modified celluloses and polysaccharide gums.

Another delivery system that is contemplated by the present inventionfor targeting lysates to the stomach while avoiding gastric emptying isknown as a hydrodynamically balanced system. This system is based oncapsules or tablets with bulk density lower than gastric fluid. Thus,the dosage form stays buoyant in the stomach. These dosage forms arecomprised of 20-75% of one or more hydrocolloids, e.g.,hydroxyethylcellulose and hydroxypropylmethylcellulose.

Other types of these devices include osmotic pressure compartmentscontaining osmotically active salts. In the present invention,dissolution of these salts by the gastric fluid pumps out the lysatecomposition. Others are based upon a floating bilayer compressed matrix.One of the layers is comprised of a hydrophilic polymer and a carbondioxide generating composition. The carbon dioxide maintains buoyancyand the other hydrophilic layer releases the drug from the matrix. Afurther method for gastric lysate targeting involves an intragastricretention shape, made of polyethylene or polyethylene blend.

The delivery systems described above may also be used in the presentinvention to target lysate compositions to the upper small intestine.However targeting to other areas of the small intestine may involveseveral additional systems. The low stomach pH and presence of gastricenzymes have led to the development of enteric coating. This coatingprotects the gastric mucosa from lysate irritation. Coating is done witha selectively insoluble substance, and protects lysates frominactivation by gastric enzymes and/or low pH.

In summary, the present invention comprises methods of administration oflysate compositions for treatment of microbial infections. Not alladministration routes are efficacious for every patient Therefore, thepresent invention comprises various methods, which require differingformulations of the lysate compositions. The formulations include thosesuitable for oral, rectal, ophthalmic (including intravitreal orintracameral), nasal, topical (including buccal and sublingual), vaginalor parenteral (including subcutaneous, transdermal, intramuscular,intravenous, intradermal, intratracheal, and epidural) administration.The formulations may conveniently be presented in unit dosage form andmay be prepared by conventional pharmaceutical techniques.

A tablet may be made by compression or molding, optionally with one ormore accessory ingredients. Compressed tablets may be prepared bycompressing, in a suitable machine, the active ingredient in afree-flowing form such as a powder or granules, optionally mixed with abinder, lubricant, inert diluent, preservative, surface active ordispersing agent. Molding, in a suitable machine, a mixture of thepowdered compound moistened with an inert liquid diluent may make moldedtablets. The tablets may be optionally coated or scored and may beformulated so as to provide a slow or controlled release of the lysatetherein.

Formulations suitable for topical administration in the mouth includelozenges comprising the ingredients in a flavored basis, usually sucroseand acacia or tragacanth; pastilles comprising the active ingredient inan inert basis such as gelatin and glycerin, or sucrose and acacia; andmouthwashes comprising the lysate in a suitable liquid carrier.

The present invention additionally comprises methods of topicaladministration. To prepare the topical composition according to thepresent invention the usual manner for preparing skin care products maybe employed. The active components are generally incorporated in adermatological acceptable carrier in conventional manner. It may bepackaged in discrete units including aerosol sprays, each containing apredetermined amount of the active ingredient, as a powder, stick, orgranules, as creams, pastes, gels, lotions, syrups, or ointments, onsponges or cotton applicators, or as a solution or a suspension in anaqueous liquid, a non-aqueous liquid, an oil-in-water emulsion, or awater-in-oil liquid emulsion. The lysates can suitably first bedissolved or dispersed in a portion of the water or another solvent orliquid to be incorporated in the composition. The composition can alsobe in the form of a so-called “wash-off” product e.g. a bath or showergel, possibly containing a delivery system for the lysates to promoteadherence to the skin during rinsing. Most preferably the product is a“leave-on” product; a product to be applied to the skin without adeliberate rinsing step soon after its application to the skin. Suchcompositions may be prepared by any of the methods of pharmacy, but allmethods include the step of bringing into association the carrier(s)with the lysate composition. In general, the compositions are preparedby uniformly and intimately admixing the active ingredient with liquidcarriers or finely divided solid carriers or both, and then, ifnecessary, shaping the product into the desired presentation. Thecomposition may packaged in any suitable manner such as in ajar, abottle, tube, roll-ball, or the like, in the conventional manner.

Formulations suitable for vaginal administration may be presented aspessaries, tamports, creams, gels, pastes, foams or spray formulationscontaining in addition to the lysates such carriers as are known in theart to be appropriate.

Formulations suitable for parenteral administration include aqueous andnon-aqueous sterile injection solutions which may contain anti-oxidants,buffers, bacteriostats and solutes which render the formulation isotonicwith the blood of the intended recipient; and aqueous and non-aqueoussterile suspensions which may include suspending agents and thickeningagents. The formulations may be presented in unit-dose or multi-dosecontainers. Extemporaneous injection solutions and suspensions may beprepared from sterile powders, granules and tablets of the kindpreviously described. Preferred unit dosage formulations are thosecontaining a daily dose or unit, daily sub-dose, as herein aboverecited, or an appropriate fraction thereof, of the administeredingredient.

The compounds may also be entrapped in microcapsules prepared, forexample, by coacervation techniques or by interfacial polymerization,for example, hydroxymethylcellulose or gelatin-microcapsules andpoly(methylmethacylate) microcapsules, respectively, in colloidal drugdelivery systems (for example, liposomes, albumin microspheres,microemulsions, nano-particles and nanocapsules) or in macroemulsions.REMINGTON'S PHARMACEUTICAL SCIENCES (A. Osol ed., 16th ed. (1980)).

In a specific embodiment, the compounds disclosed herein are formulatedas liposomes. Liposomes containing a compound of the present inventionare prepared by methods known in the art. See, e.g., U.S. Pat. Nos.5,013,556; 4,485,045; 4,544,545; WO 97/38731; Epstein et al., 82 PROC.NATL. ACAD. SCI. USA 3688 (1985); and Hwang et al., 77 PROC. NATL. ACAD.SCI. USA 4030 (1980). The compounds of the present invention can also beadministered in the form of liposome delivery systems such as smallunilamellar vesicles, large unilamellar vesicles, and multilamellarvesicles. Liposomes can be formed from a variety of phospholipids suchas cholesterol, stearylamine or phophatidylcholines.

The present invention provides stable formulations as well as preservedsolutions and formulations containing a preservative as well asmulti-use preserved formulations suitable for pharmaceutical orveterinary use, comprising at least one compound disclosed herein in apharmaceutically acceptable formulation. Formulations in accordance withthe present invention may optionally contain at least one knownpreservative.

In addition, co-administration or sequential administration of thecompounds of the present invention and other therapeutic agents may bedesirable, such as chemotherapeutic agents, immunosuppressive agents,cytokines, cytotoxic agents, nucleolytic compounds, radioactiveisotopes, receptors, and pro-drug activating enzymes, which may benaturally occurring or produced by recombinant methods. The combinedadministration includes co-administration, using separate formulationsor a single pharmaceutical formulation, and consecutive administrationin either order, wherein preferably there is a time period while both(or all) active therapeutic agents simultaneously exert their biologicalactivities.

In another embodiment, the other therapeutic agent comprises a cytokine.The term “cytokine” is a generic term for proteins released by one cellpopulation which act on another cell as intercellular mediators.Examples of such cytokines are lymphokines, monokines, and traditionalpolypeptide hormones. Included among the cytokines are growth hormonessuch as human growth hormone, N-methionyl human growth hormone, andbovine growth hormone; parathyroid hormone; thyroxine; insulin;proinsulin; relaxin; prorelaxin; glycoprotein hormones such as folliclestimulating hormone (FSH), thyroid stimulating hormone (TSH), andluteinizing hormone (LH); hepatic growth factor; fibroblast growthfactor; prolactin; placental lactogen; tumor necrosis factor-α and -β;mullerian-inhibiting substance; mouse gonadotropin-associated peptide;inhibin; activin; vascular endothelial growth factor; integrin;thrombopoietin (TPO); nerve growth factors such as NGF-β; plateletgrowth factor; transforming growth factors (TGFs) such as TGF-α andTGF-β; insulin-like growth factor-I and -II; erythropoietin (EPO);osteoinductive factors; interferons such as interferon-α, -β and -γ;colony stimulating factors (CSFs) such as macrophage-CSF (M-CSF);granulocyte-macrophage-CSF (GM-CSF); and granulocyte-CSF (GCSF);interleukins (ILs) such as IL-1, IL-la, IL-2, IL-3, IL-4, IL-S, IL-6,IL-7, IL-8, IL-9, IL-11, IL-12, IL-15; a tumor necrosis factor such asTNF-α or TNF-β; and other polypeptide factors including LIF and kitligand (KL). As used herein, the term cytokine includes proteins fromnatural sources or from recombinant cell culture and biologically activeequivalents of the native sequence cytokines.

It should be understood that in addition to the ingredients,particularly mentioned above, the formulations of the present inventionmay include other agents conventional in the art having regard to thetype of formulation in question, for example, those suitable for oraladministration may include flavoring agents.

As used herein and in the appended claims, the singular forms “a,” “an,”and “the” include plural reference unless the context clearly indicatesotherwise. Thus, for example, reference to a “compound” is a referenceto one or more such compounds and includes equivalents thereof known tothose skilled in the art, and so forth. Unless defined otherwise, alltechnical and scientific terms used herein have the same meaning ascommonly understood to one of ordinary skill in the art to which thisinvention belongs.

All publications and patents mentioned herein are incorporated herein byreference for the purpose of describing and disclosing, for example, theconstructs and methodologies that are described in the publications,which might be used in connection with the presently describedinvention. The publications discussed above and throughout the text areprovided solely for their disclosure prior to the filing date of thepresent application. Nothing herein is to be construed as an admissionthat the inventors are not entitled to antedate such disclosure byvirtue of prior invention.

It is to be understood that this invention is not limited to theparticular formulations, process steps, and materials disclosed hereinas such formulations, process steps, and materials may vary somewhat. Itis also to be understood that the terminology employed herein is usedfor the purpose of describing particular embodiments only and is notintended to be limiting since the scope of the present invention will belimited only by the appended claims and equivalents thereof.

The above disclosure generally describes the present invention. A morecomplete understanding can be obtained by reference to the followingexamples. These examples are described solely for purposes ofillustration and are not intended to limit the scope of the invention.Although specific terms have been employed herein, such terms areintended in a descriptive sense and not for purposes of limitations.

EXAMPLES Example 1 Preparation and Use of S. aureus Lysate

Strains of S. aureus bacteria were deposited with the Czech Collectionof Microorganisms (CCM) and have been assigned accession no. CCM 4992,CCM 4993, CCM 4994, CCM 4995, CCM 4996, CCM 4997, CCM 4998. Thebacteriophage used were from the group deposited with the DeutscheSammlung von Mikroorganismen and Zeilkulturen GmbH (DSM) and havingaccession nos. DSM 14614, DSM 14615, DSM 14616.

The bacteria, stored as a lyophilized culture were streaked onto tryptonagar or a similar nutrient agar medium. After 24-48 h incubation at 37°C., 50 ml trypton medium was inoculated with the bacteria grown on theagar plate. The inoculated medium was incubated at 37° C. for 18-20 hand the volume added to 500 ml medium and incubated for 1-2 h (based onOD measured at 600 nm). The phage stock was then added and the mixturewas allowed to sit at room temperature for 12-18 h. The resulting lysatewas centrifuged for 1 h at 5,000 rpm, and filtered using filters with apore size of 0.22 μm. The mixture was tested for sterility using bloodagar and phage titration using a 2-layer agar technique.

Example 2 Screening of Bacterial Strains

Bacterial strains are screened for the most common antigenic patternusing immunogenic methods such as fluorescent antibody identification,FACS selection, ELISA, Western blots, SDS gel electrophoresis or byother detection methods known to those skilled in the art. Generally,the antigenic pattern is measured by SDS gel electrophoresis. Thecomponents of the SPL are separated in a gel and silver-stained. Theantigenic pattern is determined by the presence/absence of particularbands in the gel.

Cultures of the selected bacteria were then infected by the introductionof phage homologues. The combinations of phage and bacteria werescreened for phage strains that resulted in quantitative lysis ofbacteria in a given time and strains that exhibited the most commonantigenic pattern through the above-mentioned methods. Combinations ofbacterial and phage strains that exhibit reproducible results in lysiswere selected. Combinations of one bacterial strain and different phagesresults in different antigenic combinations which can also be utilized.The selected bacterial and phage strains were preserved bylyophylization.

Example 3 Lyophilization

Phage are prepared by lysing the host bacteria resulting in 10⁰ to 10¹⁰phage particles per ml.

0.5 ml of the phage containing bacterial lysate are placed in tubes towhich 0.2 ml of skim milk has been added. The tubes are then coveredwith a sterile porous cover. The tubes are lyophilized using acryodesiccator instrument such as 30P2 or EdwardsEF03 using primarydesiccation for 4 h and secondary desiccation for 6 h. The tubes arethen sealed.

Each batch of phage containing bacterial lysate is tested for viabilityby plating the contents of one tube on a layer of host bacteria andobtaining the titer from two-fold dilutions. The titer is compared tothe starting titer prior to lyophilization.

Example 4 Stability Analysis

The stability of the lysate cultures is determined through stabilityassays which are repeated cultures and subcultures on plates and inliquid media. The stability studies are performed to select bacterialand phage strains that are most stable upon storage. The criteriameasured by methods known to those skilled in the art are the measuringof antigenic pattern by SDS gel electrophoresis; and phage virulence,excluding strains that become resistant to the phages used forinfection. The cultures are also checked for reproducibility ofantigenic patterns of lysates. Standardized inoculum of bacterial cellsby the phages results in a very reproducible process of lysis. Theresulting lysate is quantitatively and qualitatively analyzed by SDS gelelectrophoresis.

Example 5 Preparation of Immunomodulators

Biological assays (e.g. blastic transformation, major antigenic proteinsdetected by Western blots, immunomodulation, etc.) are used to identifybiologically active substances in the resultant lysates. A blastictransformation of peripheral lymphocytes is measured using individualfractions from SPL fractionated by chromatographic techniques.Activation markers or thymidine incorporation is used in the assay.Active fractions (those stimulating/inhibiting activation of peripherallymphocytes) are further fractionated and used for isolation of adesired immunomodulator. One or more of these substances are thenpurified from one or more of the resultant lysates and identified andcombinations are formed having the desired activities. Two or more ofthe resultant lysates identified are mixed together to yield the finalpolyclonal immunomodulator.

Example 6 Treatment with S. Aureus Lysates

Patients receive a subcutaneous dose 0.05 cc-0.2 cc (5 cc bottle) of thefiltrate prepared as described in Example 5 into the antebrachium(forearm). The dose is dependent on the age, clinical and immunologicalstatus of the patient. In hypersensitive patients, the doses should be0.02 cc-0.03 cc. This smaller dose is divided in half and applied intoboth antebrachiums, with the same amount administered at eachrepetition. Doses are administered every 3-5 days following theresolution of any prior local reaction. The doses are graduallyincreased in non-hypersensitive patients to 0.05 cc subcutaneously,according to individual reaction of the patient. In severe cases, thedose can be increased to 1.0 cc subcutaneous per dose. If the localreaction lasts longer then 4 days, the same dose should be repeated inthe subsequent administration.

In pediatric applications, i.e. for children aged 3-10 years, the dosageis decreased by 50% from the above regime, or may be administeredaccording to individualized reactions.

The length of the treatment is dependent on the clinical andimmunological status of the patient. Generally the lysates areadministered every 3-5 days for 3 months. This dosing regime can berepeated periodically as a booster.

In the case of milder infection or in pediatric indications the lysatecan be administered as nasal drops. The dose is 4 drops in each nostrildaily for four days and thereafter every other day. Different routes ofadministration may be combined.

Example 7 Stimulation of Metabolic Burst in Phagocytic Cells (Monocytesand Polymorphonuclear Leukocytes) in Human Blood In Vitro

Samples of blood were collected and stored with heparin. The heparinizedblood was used 2 h and 7 h after collection and the 100-microlitersamples were incubated with SPL and other negative (no addition) andpositive controls including a peptide, formyl-MetLeuPhe (FMLP)(physiological stimulator), phorbol-12-myristate-13-acetate (PMA; strongstimulator).

A bursatest kit (Orpegen Pharma) was used for detection of metabolicburst in monocytes and polymorphonuclear leukocytes, respectively. Cellsorter (FACStrak) was used to count number of positive cells and totalcells.

TABLE 1 Polymorphonuclear leukocytes Monocytes (% of positive cells) (%of positive cells) Sample 2-h sample 7-h sample 2-h sample 7-h sampleNegative 9.7 19.4 13.9 18.1 control SPL 38.2 61.7 31.3 40.2 FMLP 13.826.3 15.8 18.8 PMA 99.6 99.8 98.7 99.5Staphyloccocal lysate (SPL) induced metabolic burst in both monocytesand polymorphonuclear leukocytes in blood samples treated 2 h and 7 hafter collection. This treatment led to an increase of 2.5-3.2-fold inmonocytes and 3.6-4.3-fold in polymorphonuclear leukocytes versus thecontrol.

Example 8 Activation of Sub-Population of T Cells in Human Blood InVitro

Heparinized blood was incubated for 6 h with SPL and other negative (noaddition) and positive controls including a peptide formyl-MetLeuPhe(physiological stimulator), phorbol-12-myristate-13-acetate (PMA; strongstimulator), serum-opsonized Escherichia coli cells (E. coli-o)(gram-negative bacterium containing lipopolysaccharides), andphytohemaglutinin (PHA; T-cell stimulator). Using Fastimmune test andFACStrak with triple fluorescence, early activation (CD69+) andproduction of interpheron gamma (IFN-g) was measured in CD4+ T cells.Isotype controls were used to subtract background staining.

TABLE 2 (CD69+) & CD69+ (IFN-g+) (IFN-g+) (% of (% of (% of Samplepositive cells) positive cells) positive cells) Negative 0.56 0 0.02control PMA 76.04 3.09 0.04 fMLP 0.85 0 0 PHA 76.19 2.90 0.21 E. coli-o10.12 — 0 SPL-50 ul 14.25 0.63 0.01 SPL-100 ul 17.11 0.86 0.01 SPL-150ul 18.37 0.75 0.04SPL induced earlier and greater activation (CD69+) of CD4+ T cells thanfMLP or E. coli-o and the levels reached about 20% of those induced bymitogens PMA or PHA.

When cells producing IFN-γ were counted in the CD69+ subpopulation ofCD4+ T lymphocytes stimulated with SPL, the levels were 30% of thosestimulated with mitogens PMA or PHA.

E. coli-stimulated or fMLP-stimulated CD4+ T lymphocytes did not showany significant production of IFN-γ in the CD-69+ sub-population. Incontrast, SPL-stimulated cells showed significant production of IFN-γ inthe early-activated (CD69+) cells.

1-20. (canceled)
 21. A method of treating a pathological condition or amicrobial infection in a human or animal, comprising administering to ahuman or an animal a bacterial lysate derived from the infection of atleast one non-pathogenic bacterial strain of Staphylococcus aureus withat least one bacteriophage, wherein the at least one bacterial strain isa Staphylococcus aureus strain deposited with the Czech Collection ofMicroorganisms (CCM) having accession number CCM 4995 or CCM 4997, orboth strains CCM 4995 and CCM 4997; and the at least one bacteriophageis a bacteriophage deposited with the Deutsche Sammlung vonMikroorganismen und Zellkulturen GmBH (DSM) having accession number DSM14616.
 22. The method of claim 21, wherein the lysate further comprisesa pharmaceutical excipient.
 23. The method of claim 21, wherein thelysate further comprises an adjuvant.
 24. The method of claim 21,wherein the pathological condition or microbial infection is chronicupper respiratory disease, wound infection, mastitis, osteomyelitis,endocarditis, skin polymicrobial infections, bronchial asthma, chronicsinusitis, cystic fibrosis or acne vulgaris.
 25. The method of claim 21,wherein the lysate is administered subcutaneously, intradermally,intramuscularly, nasally, topically, or parenterally.
 26. The method ofclaim 21, wherein the lysate is administered prophylactically.
 27. Themethod of claim 21, wherein the lysate is administered more than onetime.
 28. The method of claim 21, wherein the lysate is administeredmore than one time by one or more than one administration route.
 29. Amethod of preparing a bacterial lysate, wherein the bacterial lysatecomprises, a) growing at least one bacterial strain in culture to aconcentration that is at least 1×10⁶ cells per milliliter, wherein theleast one bacterial strain is a Staphylococcus aureus (S. aureus) straindeposited with the Czech Collection of Microorganisms (CCM) havingaccession number CCM 4995 or CCM 4997, or both strains CCM 4995 and CCM4997; b) adding to the bacterial strain in culture sufficient amounts ofat least one bacteriophage to yield a concentration of at least 1×10⁸phage particles per milliliter, wherein the at least one bacteriophageis a bacteriophage deposited with the Deutsche Sammlung vonMikroorganismen und Zellkulturen GmBH (DSM) having accession number DSM14616; c) allowing infection and lysis to proceed from 3 to 48 hours;and d) sterilizing the lysate.
 30. The method of claim 29, whereinsterilizing the bacterial lysate comprises filtering the lysate througha filter with a pore size less than or equal to about 0.45 μm.
 31. Themethod of claim 30, wherein the bacterial lysate further comprises thefiltrate.
 32. The method of claim 30, wherein the bacterial lysatefurther comprises the retentate.
 33. The method of claim 30, wherein thebacterial lysate further comprises admixing fixed ratios of the filtrateand retentate.